0 1992 J. Pharm. Pharmacol.
Pharm. Pharmacol. 1992,44: 579-582 Received September 13, 1991
J.
Tetrandrine and Isotetrandrine, Two Bisbenzyltetrahydroisoquinoline Alkaloids from Menispermaceae, with Rat Uterine Smooth Muscle Relaxant Activity P I L A R D ' O C ~ N , M . A M P A R O BLAZQUEZ, A L M U D E N A BERMEJO A N D ELSA A N S E L M I
Loboratorio de Farmacognosia y Farmacodinamia, Departamento de Farmacologia, Facultad de Farmacia, Vniversidad de Valencia, Spain
Abstract-The effects of two bisbenzyltetrahydroisoquinolinealkaloids, IS, 1 'S tetrandrine and its isomer 1R, I'Sisotetrandrine, were investigated in rat isolated uterus in order to identify the mechanism of relaxant action and to study the influence of the absolute configuration on the activity of these alkaloids. Both inhibited the uterine contraction induced by high K + , acetylcholine and oxytocin. In Ca2+-freemedium, isotetrandrine relaxed the sustained contraction induced by oxytocin but tetrandrine did not. The relaxant effects of the alkaloids may be due to blockade of calcium influx through specific channels. Tetrandrine and isotetrandrine modify the calcium channel in a nonreversible manner whilst only isotetrandrine acts intracellularly. Tetrandrine shows a more specific relaxant activity as a calcium entry blocker.
Tetrandrine, a bisbenzyltetrahydroisoquinolinealkaloid isolated from the Chinese medicinal plant Sephania tetrandra, is used in China to treat angina and hypertension. Previous experiments have shown that tetrandrine exerts potent negative inotropic effects (Fang & Jiang 1986) in a variety of animal species, blocks the contraction of K+-depolarized or noradrenaline-induced contraction in rabbit aorta, shifts the cumulative dose-response curves to the right and depresses the maximal response to CaC12 in uterine smooth muscle (Fang & Jiang 1986).These pharmacological studies suggest that thiscompound may function as a Ca2+entry blocker but its precise mechanism of action remains unknown. The chemical structure of tetrandrine (Fang & Jiang 1986) shows a dimer of two benzylisoquinoline subunits condensed in a head to head, tail to tail fashion with IS, 1's stereochemistry at the chiral isoquinoline carbons. Isotetrandrine alkaloid isolated from Limaciopsis loangensis (Cave et a1 1979) differs from tetrandrine only in the absolute stereochemistry at the chiral carbon atoms (Fig. 1). In the present study we have examined the mechanism by which tetrandrine and its isomer, isotetrandrine, inhibit the contractile responses of rat uterine smooth muscle. Experiments in the presence or absence of extracellular calcium were performed to identify the mechanism of action of these alkaloids and to clarify the influence of the absolute configuration on its relaxant activity. Materials and Methods Preparation of uterine horns
Female Wistar rats, 150-200 g, were given oestradiol benzoate ( 5 mg kg-I); 24 h later they were killed by a blow on the head and exsanguinated. One uterine horn was removed and mounted in a 10 mL organ-bath chamber filled with Correspondence:P. D'Ocon, Laboratorio de Farmacognosia y Famacodinamia, Departamento de Farmacologia, Facultad de Fama$a, Universidad de Valencia, Avda. Blasco Ibaiiez 13,46010Valenaa, Spain.
OCH,
Tetrandrine
Tetrandrine
FIG.1 . Chemical structure of tendrandrine and isotetrandrine.
physiological solution bubbled with a mixture of 95% 025% c02, 31°C. Experimental procedure K+-depolarized uterus. The organ was immersed in Jalon-
Ringer solution and equilibrated for 20 min under a resting tension of 1 g and a prolonged tonic contraction was obtained (KCI,). Two experimental designs were applied. In one series of experiments, three contractile responses to KCI were induced and alkaloids M) were added 15 min before the second addition of depolarizing solution (KClb). After washing, another addition of depolarizing solution induced a contractile response (KCL). In another series, alkaloids were added in cumulative amounts (10 min interval between doses) when the second plateau of K+-contraction (KClb) was reached (cumulative amounts 10a6-3.33x M). After washing, further addition of depolarizing solution induced a contractile response (KCL). In order to investigate the effect of the alkaloids on /3adrenoceptors, this assay was run in the presence and absence of propranolol M).
580
PILAR D'OCON ET AL
Acetylcholine-induced contraction. A uterine horn was incubated in Jalon-Ringer solution with a resting tension of 1 g for 20 min. Acetylcholine (ACh, M) was added, which induced an initial phasic contraction followed by a plateau with small rhythmic contractions. Two experimental procedures were performed as above. In the first, the alkaloids M) were added 15 min before the second addition of acetylcholine (AChb) and after washing, a third contraction induced by this agonist was obtained (AChJ. In the second, cumulative amounts of the alkaloids were added when the second contractile response to the agonist (AChb) was reached (cumulative amounts 3.33 x M). After washing, another addition of acetylcholine induced a contractile response (ACh,). Oxytocin-induced rhythmic contractions. A uterine horn was incubated in Locke-Ringer solution with a resting tension of 1 g for 20 min. Oxytocin (0.01 unit mL-l) was added, inducing rhythmic contractions. When these were stable the alkaloids were added cumulatively to the organ bath. Oxytoxin-induced Ca2+-freecontractions. A uterine horn was equilibrated for 1 h in Locke-Ringer solutions under a resting tension of 0.5 g. The solution was then replaced by Ca2 -free solution containing 3 mM EDTA and incubation was continued for 50 min. Subsequently, the solution was replaced by Ca2+-freesolution containing 1 mM EDTA and the uterus was incubated for 20 to 30 min. Sustained contractile responses to oxytocin (0.01 unit mL-l) were obtained and cumulative amounts of alkaloids were added. Isometric responses were measured using a recorder (Philips PM 8222) with an amplifier (8805C HP) and a force displacement transducer (Gould Statham UC2). Solutions The following solutions were used: Jalon-Ringer (mM): NaCl 154, KCI 5.63, CaCI20.648, NaHC035.95 and glucose 2.77; depolarizing Jalon-Ringer (mM):NaCI 103.3, KCI 56.3 CaClz 0.648, NaHC03 5.95 and glucose 2.77; Locke-Ringer (mM): NaCll54, KCI 5.63, CaC122.16, MgCI22.10, NaHC03 5.95 and glucose 5.55; Ca2+-free Locke-Ringer: as for Locke-Ringer except for the omission of CaC12and addition of EDTA 3 or 1 mM. Drugs Oxytocin and EDTA were purchased from Sigma Chemical Company. Tetrandrine was a generous gift from Dr Fang. Isotetrandrine was isolated by Cave et a1 (1979). All drugs were dissolved in distilled water. All solutions were prepared daily and the pH 7 was confirmed. All other chemicals used were of analytical grade. Statistical analysis Relaxations are expressed as a percentage of the maximum tension obtained by agonist addition. Em,, represents the maximal relaxation obtained after addition of the higher dose of each compound tested. A regression of response against -log C of test compound was performed by the least squares method for each preparation. The concentration needed to produce 50% inhibition (IC50) was obtained by
linear regression of all points between 20-80% of the maximal response to the alkaloid tested. Results are expressed as the mean+s.e.m. of 5 or more preparations (n) obtained from different animals. Statistical significance of differences between the means was obtained using Student's t-test for unpaired data. P-values < 0.05 were considered to represent significant differences. Results Relaxant effects of alkaloids on K+-depolarizedrat uterus The three successive additions of KCI induced contractile responses of similar magnitude. When alkaloids M) were added 15 min before the second K+-induced contraction (KClb), a lower contractile response was obtained. The phasic contraction to KCI in the presence of tetrandrine or isotetrandrine was significantly diminished in comparison with the contraction obtained when alkaloids were not present (Fig. 2A). The tonic component was completely inhibited. After washing, a new addition of KCI (KCL) did not reproduce the initial contraction. The addition of tetrandrine and isotetrandrine when the plateau of the second K+-induced contraction was reached produced concentration-dependent relaxation. Parameters of dose-response curves of relaxation are presented in Table 1. After washing the uterus, a new addition of KCl (56.3 mM) produced a contraction plateau (KCI,) that was significantly different from the first. In the presence of propranolol M), the alkaloids completely relaxed the KC1-induced contraction and the IC50 was not different from that obtained in the absence of propranolol (ICSO of tetrandrine: 434 f 13.3 p ~ n,= 5; IC50 of isotetrandrine: 45.3 5.6 p ~ n ,= 5). This concentration of
A KCI
A Alk
A KCL
A KCI
B
15'
h
FIG.2. Effect of alkaloids M) added 15 rnin before the second M). addition of (A) depolarizingsolution and (B) acetylcholine(
UTERINE RELAXANT ACTIVITY FROM TETRANDRINE AND ISOTETRANDRINE
Table 1. Parameters of dose-response curves for the relaxation induced by cumulative doses of tetrandrine and isotetrandrine in rat KC1-depolarizeduterus, acetylcholine (ACh) contraction, ox tocin (OTca(+))(0.01 units mL-I) contractions and oxytocin (OTc+,) (0.01unit mL- 7) Ca2+-freecontractions. Tetrandrine IC50 (lo-'
La,(YO) KCI
ACh OTca(+) OTca(- )
l O O 5 + 1.0 100.1 k 1.3 945k3.4
Isotetrandrine M)
3.2k0.5 2.8f0.5 4.5f0.9
n
5 9 6
%ax
(%)
106.3+4.2 91.7+1.4 83,854.2 89.1 2.2
+
IC50
(w M)
2.7 f0.3 3.9k0.9 4.9k1.5 3 3 + 1.1
n 5 5 4 6
Values are mean & s.e.m.
propranold has previously been shown to block the relaxant action of isoprenaline. Relaxant eflects of alkaloids on acetylcholine-induced contraction of rat uterus When the uterus was preloaded with tetrandrine or isotetrandine M), 15 min before the addition of acetylcholine M) (AChb), inhibition of the phasic peak contraction with respect to the contractile response obtained in the absence of the alkaloids was observed. The rhythmic contractions were abolished only by tetrandrine (Fig. 2B). After washing the uterus, a new addition of acetylcholine M) (AChJ did not reproduce the first contraction. Addition of tetrandrine and isotetrandrine during the second plateau of contraction by acetylcholine (AChb) produced dose-dependent relaxations; hence, dose-response (relaxation) curves were constructed by addition of cumulative doses of alkaloids (Table 1). After washing the uterus, a new addition of acetylcholine (AChJ M) produced a contraction that was significantly different from the first. Modijication of uterine response to oxytocin by the alkaloids The addition of oxytocin units mL-l) to the uterine -Log concn (M)
5
6 O1
=
- -
4
3
a
100 3. Dose response curves of relaxation to alkaloids in uterus Previously contracted with oxytocin (0.01 units mL-l) incubated in %'+-free EDTA-containing solution. 0 Tetrandrine (n = 51, 'Sotetrandrine (n = 5). Bars represent s.e. of n experiments. FIG.
horn incubated in Locke-Ringer solution induced rhythmic contractile responses of stable frequency and amplitude (3668.40f447.32 mg, n = 5). The addition of cumulative concentrations (10-6-3.33 x M) ofboth alkaloids diminished the frequency and amplitude of the contraction in a concentration-dependent manner. IC50 values obtained for each alkaloid were similar to those obtained for experiments with acetylcholine of KCI (Table 1). Effects of alkaloids on contractile response of uterus to oxytocin in Ca2+-freemedium The sustained contractions (343.0f 1.7 mg, n = 5) induced by oxytocin (0.01 units mL-l) after 20 min in Ca2+-free EDTA-containing solution were not modified when cumulative doses (10-6-3.33 x M) of tetrandrine were added (n=5) (Fig. 3). In contrast, cumulative doses M) of isotetrandrine produced concentration3.33 x dependent relaxation (Table 1).
Discussion The results show that tetrandrine and its isomer, isotetrandrine, are able to inhibit several models of agonist-induced contractions in smooth muscle. In the presence of extracellular Ca2+, both alkaloids inhibited, in a concentration-dependent manner, the contractile response induced by depolarization with acetylcholine or oxytocin, when they were added before or during the contractile plateau elicited by each agonist. Examination of characteristic parameters of dose-response curves of relaxation indicates that Emaxwas similar in all cases and IC50 was not significantly different. The relaxant action exhibited by tetrandrine and isotetrandrine was similar in the presence or absence of propranolol. This indicates that the relaxation induced by the alkaloids cannot be attributed to a B-stimulating action. These results agree with those of previous studies with tetrandrine in rabbit aorta and coronary arteries (Fang & Jiang 1986). The response to KCI includes a biphasic contraction consisting of an early phasic component and delayed tonic component that is the result of increased Ca2+influx through voltage-sensitive calcium channels (Godfraind & Kaba 1972). The phasic contraction was lower in the presence of the alkaloids tested but the tonic contraction was abolished when the uterus was preincubated with the alkaloid or when cumulative concentrations of tetrandrine or isotetrandrine were added after the sustained contractile plateau induced by KC1 was reached. It is interesting to note that contractile
582
PILAR D ’ O C ~ NET AL
responses to KCl after removal of the alkaloid from the medium did not recover in any of the experimental procedures. These results indicate that the alkaloids affect KClsensitiveCa2+-channelsby a mechanism that is not reversible by washing. The mechanism underlying the rise in cytoplasmic Ca2+ induced by acetylcholine and oxytocin is complex and probably involves both voltage-dependent and receptoroperated calcium channels (Bolton 1979; Edwards et a1 1986; Savineau & Mironneau 1990), as well as release of intracellular Ca2+ions (Villar et a1 1986; Anselmi et a1 1987; D’Ocon et a1 1987a, b). Tetrandrine and isotetrandrine relax uterine contraction induced by both agonists in a dose-dependent manner. When alkaloids were added before the second addition of acetylcholine a lower but sustained contractile response was obtained, unlike KC1-induced contraction in the presence of the alkaloids. These results suggest a more specific action on the contractile response induced by KC1. In order to determine the possible action of alkaloids on intracellular Ca2+,experiments were designed in Ca2+-free medium. Under these conditions only oxytocin can promote a sustained contraction (Anselmi et a1 1987; DOcbn et a1 1987a). Acetylcholine induces a slight phasic contraction (DOcbn et a1 1987b) and KCI does not elicit uterine contractile responses ( W a r et a1 1986). In Ca2+-freeEDTAcontaining solution the maintained contraction evoked by oxytocin is mediated by the release of Ca 2+ from intracellular calcium stores through activation of a specific receptor (Anselmi et a1 1987). The present results indicate that tetrandrine has no effect on the release of Ca2+ from an intracellular store or stores sensitive to oxytocin but since isotetrandrine relaxed oxytocin contractions in Ca2 -free solution in a dose dependent manner, it may act intracellularly. The different behaviour of the two alkaloids on uterine contraction promoted by the release of intracellular Ca2+ may be related to the absolute configuration at the chiral centres. The alkaloid with absolute configuration IS, 1’s (tetrandrine) exerts a selective inhibitory action on the calcium influx from the extracellular medium without changing the intracellular distribution of this ion. The alkaloid with absolute configuration 1R, 1‘s (isotetrandrine) has a non-specific effect on extracellular influx and intracellular calcium levels. These observations suggest the existence of a stereospecific receptor site at the intracellular level; similar observations were made in previous studies on the antioquine series and its derivatives (DOc6n et a1 1989; Ivorra et al, 1992) or papaverine and its derivatives (Anselmi et al, 1992). When these same experimental procedures are applied using nifedipine as a relaxant of uterine contraction, the results resemble those obtained with tetrandrine (D’Ocon et a1 1991). These results confirm those of King et a1 (1988) who showed that tetrandrine interacts directly at the benzothiazepine-binding site of the Ca2+-channeland modulates ligand binding allosterically at other receptors in this complex. More recently, Liu et a1 (1991) demonstrated that tetrandrine is a calcium channel antagonist which inhibits both T and L channel currents in ventricular cells. The present findings suggest that the relaxant effects of tetrandrine and isotetrandrine may be due to blockade of +
calcium movements across the cell membrane through specific channels. Tetrandrine and its isomer, isotetrandrine, modify the calcium channel activity in a permanent manner but only isotetrandrine acts intracellularly. Tetrandrine shows a more specific relaxant activity and inhibits calcium entry.
Acknowledgement The authors thank Professor Dr D. Fang and Dr D. Cortes for the donation of tetrandrine and isotetrandrine respectively. This work was supported by Grant No. PB86-0462 from the Spanish Comision Interministerial de Ciencia y Tecnologia.
References Anselmi, E., D’Ocon, M. P., Villar, A. (1987) A comparison of uterine contraction induced by PGEl and oxytocin in Ca-free solution. Prostaglandins 34: 351-358 Anselmi, E., Fayos, G., Blasco, R., Candenas, L., Cortes, D., D’Ocon, P. (1992) Selective inhibition ofcalcium entry induced by benzylisoquinolinesin rat smooth muscle. J. Pharm. Pharmacol. 44:331-343 Bolton, T.B. (1979) Mechanisms of action of transmitters and other substances on smooth muscle. Physiol. Rev. 59: 606-718 Cavk, A., Leboeuf, R., Hocquemiller, R., Bouquet, A., Fournet, A. (1979) Alcaliodes de Limaciopsis loangemis. Planta Med. 35: 31-41
D’Ocon, M. P., Anselmi, E., War, A. (1987a) Effects ofmagnesium chloride on thecontractile response of uterus to several agonists in Ca-free solution. J. Pharm. Pharmacol. 39: 4 4 4 4 8 DOcon, M. P., Anselmi, E., Villar, A. (1987b) Role of stored Ca in contractions of rat uterus induced by acetylcholine: effects of preincubation with Mg-free solution. Arch. Int. Phannacodyn. Ther. 286: 162-170 DOcon, M. P., Candenas, M. L., Anselmi, E.,Zafra-Polo, M.C., Cortes, D. (1989) Antioquine: a new bisbenzylisoquinoleine alkaloid with calcium antagonist activity. Ibid. 297 205-216 D’Ocon, M. P., Blasco, R., Candenas, L., Ivorra, D., Lopez, S., Villaverde, C., Castedo, L.,Cortes, D. (1991) Relaxant effects of cularines and isocrasifoline on smooth muscle contractions: interactions with calcium entry through POC,. Eur. J. Pharmacol. 196: 183-187
Edwards, D., Good, D. M., Granger, S. E., Hollingsworth, M., Robson, A., Small, R. C., Weston, A. H. (1986) The spasmogenic action of oxytocin in the rat uterus-comparison with other agonists. Br. J. Pharmacol. 88: 899-908 Fang, D. C. Jiang, M. X. (1986) Studies on tetrandrine calcium antagonistic action. Chin. Med. J. 99: 638-644 Godfraind, T., Kaba, A. (1972) The role of calcium in the action of drugs on vascular smooth muscle. Arch. Int. Pharmacodyn. Ther. 196: 35-49
Ivorra, M. D., Cercos, A., Zafra-Polo, M. C., Pkrez-Prieto, J., Saez, J.. Cortes, D., DOcon, M. P. (1992) Selectivechiral inhibition of calcium entry promoted by bisbenzyltetrahydroisoquinolinesin rat uterus. Eur. J. Pharmacol. (In press) King, V. F., Garcia, M. L., Himmel, D., Reuben, J. P., Lam, Y.T., Pan, J., Han, G., Kaczorowski, J. (1988) Interaction of tetrandrine with slowly inactivating calcium channels. J. Biol. Chem. 263: 2238-2244
Liu, Q. Y.,Karpinski, E., Pang, P. K. T. (1991) Tetrandrine inhibits both T and L calcium channel currents in ventricular cells. Abstract Book of 5th International Symposium on Calcium Antagonists: Pharmacology and Clinical Research, p. 94 Savineau,J. P., Mironneau, J. (1990) Caffeine acting on pregnant rat myometrium: analysis of its relaxant action and its failure to release Ca from intracellular stores. Br. J. Pharmacol. 99: 261-267 Villar, A., DOcon, P., Anselmi, E. (1986) Role of intracellula calcium stores in the contractile response of uterus to several agonists. J. Pharmacol. 17: 541-546
Pharm. Pharmacol. 1992,44: 579-582 Received September 13, 1991
J.
Tetrandrine and Isotetrandrine, Two Bisbenzyltetrahydroisoquinoline Alkaloids from Menispermaceae, with Rat Uterine Smooth Muscle Relaxant Activity P I L A R D ' O C ~ N , M . A M P A R O BLAZQUEZ, A L M U D E N A BERMEJO A N D ELSA A N S E L M I
Loboratorio de Farmacognosia y Farmacodinamia, Departamento de Farmacologia, Facultad de Farmacia, Vniversidad de Valencia, Spain
Abstract-The effects of two bisbenzyltetrahydroisoquinolinealkaloids, IS, 1 'S tetrandrine and its isomer 1R, I'Sisotetrandrine, were investigated in rat isolated uterus in order to identify the mechanism of relaxant action and to study the influence of the absolute configuration on the activity of these alkaloids. Both inhibited the uterine contraction induced by high K + , acetylcholine and oxytocin. In Ca2+-freemedium, isotetrandrine relaxed the sustained contraction induced by oxytocin but tetrandrine did not. The relaxant effects of the alkaloids may be due to blockade of calcium influx through specific channels. Tetrandrine and isotetrandrine modify the calcium channel in a nonreversible manner whilst only isotetrandrine acts intracellularly. Tetrandrine shows a more specific relaxant activity as a calcium entry blocker.
Tetrandrine, a bisbenzyltetrahydroisoquinolinealkaloid isolated from the Chinese medicinal plant Sephania tetrandra, is used in China to treat angina and hypertension. Previous experiments have shown that tetrandrine exerts potent negative inotropic effects (Fang & Jiang 1986) in a variety of animal species, blocks the contraction of K+-depolarized or noradrenaline-induced contraction in rabbit aorta, shifts the cumulative dose-response curves to the right and depresses the maximal response to CaC12 in uterine smooth muscle (Fang & Jiang 1986).These pharmacological studies suggest that thiscompound may function as a Ca2+entry blocker but its precise mechanism of action remains unknown. The chemical structure of tetrandrine (Fang & Jiang 1986) shows a dimer of two benzylisoquinoline subunits condensed in a head to head, tail to tail fashion with IS, 1's stereochemistry at the chiral isoquinoline carbons. Isotetrandrine alkaloid isolated from Limaciopsis loangensis (Cave et a1 1979) differs from tetrandrine only in the absolute stereochemistry at the chiral carbon atoms (Fig. 1). In the present study we have examined the mechanism by which tetrandrine and its isomer, isotetrandrine, inhibit the contractile responses of rat uterine smooth muscle. Experiments in the presence or absence of extracellular calcium were performed to identify the mechanism of action of these alkaloids and to clarify the influence of the absolute configuration on its relaxant activity. Materials and Methods Preparation of uterine horns
Female Wistar rats, 150-200 g, were given oestradiol benzoate ( 5 mg kg-I); 24 h later they were killed by a blow on the head and exsanguinated. One uterine horn was removed and mounted in a 10 mL organ-bath chamber filled with Correspondence:P. D'Ocon, Laboratorio de Farmacognosia y Famacodinamia, Departamento de Farmacologia, Facultad de Fama$a, Universidad de Valencia, Avda. Blasco Ibaiiez 13,46010Valenaa, Spain.
OCH,
Tetrandrine
Tetrandrine
FIG.1 . Chemical structure of tendrandrine and isotetrandrine.
physiological solution bubbled with a mixture of 95% 025% c02, 31°C. Experimental procedure K+-depolarized uterus. The organ was immersed in Jalon-
Ringer solution and equilibrated for 20 min under a resting tension of 1 g and a prolonged tonic contraction was obtained (KCI,). Two experimental designs were applied. In one series of experiments, three contractile responses to KCI were induced and alkaloids M) were added 15 min before the second addition of depolarizing solution (KClb). After washing, another addition of depolarizing solution induced a contractile response (KCL). In another series, alkaloids were added in cumulative amounts (10 min interval between doses) when the second plateau of K+-contraction (KClb) was reached (cumulative amounts 10a6-3.33x M). After washing, further addition of depolarizing solution induced a contractile response (KCL). In order to investigate the effect of the alkaloids on /3adrenoceptors, this assay was run in the presence and absence of propranolol M).
580
PILAR D'OCON ET AL
Acetylcholine-induced contraction. A uterine horn was incubated in Jalon-Ringer solution with a resting tension of 1 g for 20 min. Acetylcholine (ACh, M) was added, which induced an initial phasic contraction followed by a plateau with small rhythmic contractions. Two experimental procedures were performed as above. In the first, the alkaloids M) were added 15 min before the second addition of acetylcholine (AChb) and after washing, a third contraction induced by this agonist was obtained (AChJ. In the second, cumulative amounts of the alkaloids were added when the second contractile response to the agonist (AChb) was reached (cumulative amounts 3.33 x M). After washing, another addition of acetylcholine induced a contractile response (ACh,). Oxytocin-induced rhythmic contractions. A uterine horn was incubated in Locke-Ringer solution with a resting tension of 1 g for 20 min. Oxytocin (0.01 unit mL-l) was added, inducing rhythmic contractions. When these were stable the alkaloids were added cumulatively to the organ bath. Oxytoxin-induced Ca2+-freecontractions. A uterine horn was equilibrated for 1 h in Locke-Ringer solutions under a resting tension of 0.5 g. The solution was then replaced by Ca2 -free solution containing 3 mM EDTA and incubation was continued for 50 min. Subsequently, the solution was replaced by Ca2+-freesolution containing 1 mM EDTA and the uterus was incubated for 20 to 30 min. Sustained contractile responses to oxytocin (0.01 unit mL-l) were obtained and cumulative amounts of alkaloids were added. Isometric responses were measured using a recorder (Philips PM 8222) with an amplifier (8805C HP) and a force displacement transducer (Gould Statham UC2). Solutions The following solutions were used: Jalon-Ringer (mM): NaCl 154, KCI 5.63, CaCI20.648, NaHC035.95 and glucose 2.77; depolarizing Jalon-Ringer (mM):NaCI 103.3, KCI 56.3 CaClz 0.648, NaHC03 5.95 and glucose 2.77; Locke-Ringer (mM): NaCll54, KCI 5.63, CaC122.16, MgCI22.10, NaHC03 5.95 and glucose 5.55; Ca2+-free Locke-Ringer: as for Locke-Ringer except for the omission of CaC12and addition of EDTA 3 or 1 mM. Drugs Oxytocin and EDTA were purchased from Sigma Chemical Company. Tetrandrine was a generous gift from Dr Fang. Isotetrandrine was isolated by Cave et a1 (1979). All drugs were dissolved in distilled water. All solutions were prepared daily and the pH 7 was confirmed. All other chemicals used were of analytical grade. Statistical analysis Relaxations are expressed as a percentage of the maximum tension obtained by agonist addition. Em,, represents the maximal relaxation obtained after addition of the higher dose of each compound tested. A regression of response against -log C of test compound was performed by the least squares method for each preparation. The concentration needed to produce 50% inhibition (IC50) was obtained by
linear regression of all points between 20-80% of the maximal response to the alkaloid tested. Results are expressed as the mean+s.e.m. of 5 or more preparations (n) obtained from different animals. Statistical significance of differences between the means was obtained using Student's t-test for unpaired data. P-values < 0.05 were considered to represent significant differences. Results Relaxant effects of alkaloids on K+-depolarizedrat uterus The three successive additions of KCI induced contractile responses of similar magnitude. When alkaloids M) were added 15 min before the second K+-induced contraction (KClb), a lower contractile response was obtained. The phasic contraction to KCI in the presence of tetrandrine or isotetrandrine was significantly diminished in comparison with the contraction obtained when alkaloids were not present (Fig. 2A). The tonic component was completely inhibited. After washing, a new addition of KCI (KCL) did not reproduce the initial contraction. The addition of tetrandrine and isotetrandrine when the plateau of the second K+-induced contraction was reached produced concentration-dependent relaxation. Parameters of dose-response curves of relaxation are presented in Table 1. After washing the uterus, a new addition of KCl (56.3 mM) produced a contraction plateau (KCI,) that was significantly different from the first. In the presence of propranolol M), the alkaloids completely relaxed the KC1-induced contraction and the IC50 was not different from that obtained in the absence of propranolol (ICSO of tetrandrine: 434 f 13.3 p ~ n,= 5; IC50 of isotetrandrine: 45.3 5.6 p ~ n ,= 5). This concentration of
A KCI
A Alk
A KCL
A KCI
B
15'
h
FIG.2. Effect of alkaloids M) added 15 rnin before the second M). addition of (A) depolarizingsolution and (B) acetylcholine(
UTERINE RELAXANT ACTIVITY FROM TETRANDRINE AND ISOTETRANDRINE
Table 1. Parameters of dose-response curves for the relaxation induced by cumulative doses of tetrandrine and isotetrandrine in rat KC1-depolarizeduterus, acetylcholine (ACh) contraction, ox tocin (OTca(+))(0.01 units mL-I) contractions and oxytocin (OTc+,) (0.01unit mL- 7) Ca2+-freecontractions. Tetrandrine IC50 (lo-'
La,(YO) KCI
ACh OTca(+) OTca(- )
l O O 5 + 1.0 100.1 k 1.3 945k3.4
Isotetrandrine M)
3.2k0.5 2.8f0.5 4.5f0.9
n
5 9 6
%ax
(%)
106.3+4.2 91.7+1.4 83,854.2 89.1 2.2
+
IC50
(w M)
2.7 f0.3 3.9k0.9 4.9k1.5 3 3 + 1.1
n 5 5 4 6
Values are mean & s.e.m.
propranold has previously been shown to block the relaxant action of isoprenaline. Relaxant eflects of alkaloids on acetylcholine-induced contraction of rat uterus When the uterus was preloaded with tetrandrine or isotetrandine M), 15 min before the addition of acetylcholine M) (AChb), inhibition of the phasic peak contraction with respect to the contractile response obtained in the absence of the alkaloids was observed. The rhythmic contractions were abolished only by tetrandrine (Fig. 2B). After washing the uterus, a new addition of acetylcholine M) (AChJ did not reproduce the first contraction. Addition of tetrandrine and isotetrandrine during the second plateau of contraction by acetylcholine (AChb) produced dose-dependent relaxations; hence, dose-response (relaxation) curves were constructed by addition of cumulative doses of alkaloids (Table 1). After washing the uterus, a new addition of acetylcholine (AChJ M) produced a contraction that was significantly different from the first. Modijication of uterine response to oxytocin by the alkaloids The addition of oxytocin units mL-l) to the uterine -Log concn (M)
5
6 O1
=
- -
4
3
a
100 3. Dose response curves of relaxation to alkaloids in uterus Previously contracted with oxytocin (0.01 units mL-l) incubated in %'+-free EDTA-containing solution. 0 Tetrandrine (n = 51, 'Sotetrandrine (n = 5). Bars represent s.e. of n experiments. FIG.
horn incubated in Locke-Ringer solution induced rhythmic contractile responses of stable frequency and amplitude (3668.40f447.32 mg, n = 5). The addition of cumulative concentrations (10-6-3.33 x M) ofboth alkaloids diminished the frequency and amplitude of the contraction in a concentration-dependent manner. IC50 values obtained for each alkaloid were similar to those obtained for experiments with acetylcholine of KCI (Table 1). Effects of alkaloids on contractile response of uterus to oxytocin in Ca2+-freemedium The sustained contractions (343.0f 1.7 mg, n = 5) induced by oxytocin (0.01 units mL-l) after 20 min in Ca2+-free EDTA-containing solution were not modified when cumulative doses (10-6-3.33 x M) of tetrandrine were added (n=5) (Fig. 3). In contrast, cumulative doses M) of isotetrandrine produced concentration3.33 x dependent relaxation (Table 1).
Discussion The results show that tetrandrine and its isomer, isotetrandrine, are able to inhibit several models of agonist-induced contractions in smooth muscle. In the presence of extracellular Ca2+, both alkaloids inhibited, in a concentration-dependent manner, the contractile response induced by depolarization with acetylcholine or oxytocin, when they were added before or during the contractile plateau elicited by each agonist. Examination of characteristic parameters of dose-response curves of relaxation indicates that Emaxwas similar in all cases and IC50 was not significantly different. The relaxant action exhibited by tetrandrine and isotetrandrine was similar in the presence or absence of propranolol. This indicates that the relaxation induced by the alkaloids cannot be attributed to a B-stimulating action. These results agree with those of previous studies with tetrandrine in rabbit aorta and coronary arteries (Fang & Jiang 1986). The response to KCI includes a biphasic contraction consisting of an early phasic component and delayed tonic component that is the result of increased Ca2+influx through voltage-sensitive calcium channels (Godfraind & Kaba 1972). The phasic contraction was lower in the presence of the alkaloids tested but the tonic contraction was abolished when the uterus was preincubated with the alkaloid or when cumulative concentrations of tetrandrine or isotetrandrine were added after the sustained contractile plateau induced by KC1 was reached. It is interesting to note that contractile
582
PILAR D ’ O C ~ NET AL
responses to KCl after removal of the alkaloid from the medium did not recover in any of the experimental procedures. These results indicate that the alkaloids affect KClsensitiveCa2+-channelsby a mechanism that is not reversible by washing. The mechanism underlying the rise in cytoplasmic Ca2+ induced by acetylcholine and oxytocin is complex and probably involves both voltage-dependent and receptoroperated calcium channels (Bolton 1979; Edwards et a1 1986; Savineau & Mironneau 1990), as well as release of intracellular Ca2+ions (Villar et a1 1986; Anselmi et a1 1987; D’Ocon et a1 1987a, b). Tetrandrine and isotetrandrine relax uterine contraction induced by both agonists in a dose-dependent manner. When alkaloids were added before the second addition of acetylcholine a lower but sustained contractile response was obtained, unlike KC1-induced contraction in the presence of the alkaloids. These results suggest a more specific action on the contractile response induced by KC1. In order to determine the possible action of alkaloids on intracellular Ca2+,experiments were designed in Ca2+-free medium. Under these conditions only oxytocin can promote a sustained contraction (Anselmi et a1 1987; DOcbn et a1 1987a). Acetylcholine induces a slight phasic contraction (DOcbn et a1 1987b) and KCI does not elicit uterine contractile responses ( W a r et a1 1986). In Ca2+-freeEDTAcontaining solution the maintained contraction evoked by oxytocin is mediated by the release of Ca 2+ from intracellular calcium stores through activation of a specific receptor (Anselmi et a1 1987). The present results indicate that tetrandrine has no effect on the release of Ca2+ from an intracellular store or stores sensitive to oxytocin but since isotetrandrine relaxed oxytocin contractions in Ca2 -free solution in a dose dependent manner, it may act intracellularly. The different behaviour of the two alkaloids on uterine contraction promoted by the release of intracellular Ca2+ may be related to the absolute configuration at the chiral centres. The alkaloid with absolute configuration IS, 1’s (tetrandrine) exerts a selective inhibitory action on the calcium influx from the extracellular medium without changing the intracellular distribution of this ion. The alkaloid with absolute configuration 1R, 1‘s (isotetrandrine) has a non-specific effect on extracellular influx and intracellular calcium levels. These observations suggest the existence of a stereospecific receptor site at the intracellular level; similar observations were made in previous studies on the antioquine series and its derivatives (DOc6n et a1 1989; Ivorra et al, 1992) or papaverine and its derivatives (Anselmi et al, 1992). When these same experimental procedures are applied using nifedipine as a relaxant of uterine contraction, the results resemble those obtained with tetrandrine (D’Ocon et a1 1991). These results confirm those of King et a1 (1988) who showed that tetrandrine interacts directly at the benzothiazepine-binding site of the Ca2+-channeland modulates ligand binding allosterically at other receptors in this complex. More recently, Liu et a1 (1991) demonstrated that tetrandrine is a calcium channel antagonist which inhibits both T and L channel currents in ventricular cells. The present findings suggest that the relaxant effects of tetrandrine and isotetrandrine may be due to blockade of +
calcium movements across the cell membrane through specific channels. Tetrandrine and its isomer, isotetrandrine, modify the calcium channel activity in a permanent manner but only isotetrandrine acts intracellularly. Tetrandrine shows a more specific relaxant activity and inhibits calcium entry.
Acknowledgement The authors thank Professor Dr D. Fang and Dr D. Cortes for the donation of tetrandrine and isotetrandrine respectively. This work was supported by Grant No. PB86-0462 from the Spanish Comision Interministerial de Ciencia y Tecnologia.
References Anselmi, E., D’Ocon, M. P., Villar, A. (1987) A comparison of uterine contraction induced by PGEl and oxytocin in Ca-free solution. Prostaglandins 34: 351-358 Anselmi, E., Fayos, G., Blasco, R., Candenas, L., Cortes, D., D’Ocon, P. (1992) Selective inhibition ofcalcium entry induced by benzylisoquinolinesin rat smooth muscle. J. Pharm. Pharmacol. 44:331-343 Bolton, T.B. (1979) Mechanisms of action of transmitters and other substances on smooth muscle. Physiol. Rev. 59: 606-718 Cavk, A., Leboeuf, R., Hocquemiller, R., Bouquet, A., Fournet, A. (1979) Alcaliodes de Limaciopsis loangemis. Planta Med. 35: 31-41
D’Ocon, M. P., Anselmi, E., War, A. (1987a) Effects ofmagnesium chloride on thecontractile response of uterus to several agonists in Ca-free solution. J. Pharm. Pharmacol. 39: 4 4 4 4 8 DOcon, M. P., Anselmi, E., Villar, A. (1987b) Role of stored Ca in contractions of rat uterus induced by acetylcholine: effects of preincubation with Mg-free solution. Arch. Int. Phannacodyn. Ther. 286: 162-170 DOcon, M. P., Candenas, M. L., Anselmi, E.,Zafra-Polo, M.C., Cortes, D. (1989) Antioquine: a new bisbenzylisoquinoleine alkaloid with calcium antagonist activity. Ibid. 297 205-216 D’Ocon, M. P., Blasco, R., Candenas, L., Ivorra, D., Lopez, S., Villaverde, C., Castedo, L.,Cortes, D. (1991) Relaxant effects of cularines and isocrasifoline on smooth muscle contractions: interactions with calcium entry through POC,. Eur. J. Pharmacol. 196: 183-187
Edwards, D., Good, D. M., Granger, S. E., Hollingsworth, M., Robson, A., Small, R. C., Weston, A. H. (1986) The spasmogenic action of oxytocin in the rat uterus-comparison with other agonists. Br. J. Pharmacol. 88: 899-908 Fang, D. C. Jiang, M. X. (1986) Studies on tetrandrine calcium antagonistic action. Chin. Med. J. 99: 638-644 Godfraind, T., Kaba, A. (1972) The role of calcium in the action of drugs on vascular smooth muscle. Arch. Int. Pharmacodyn. Ther. 196: 35-49
Ivorra, M. D., Cercos, A., Zafra-Polo, M. C., Pkrez-Prieto, J., Saez, J.. Cortes, D., DOcon, M. P. (1992) Selectivechiral inhibition of calcium entry promoted by bisbenzyltetrahydroisoquinolinesin rat uterus. Eur. J. Pharmacol. (In press) King, V. F., Garcia, M. L., Himmel, D., Reuben, J. P., Lam, Y.T., Pan, J., Han, G., Kaczorowski, J. (1988) Interaction of tetrandrine with slowly inactivating calcium channels. J. Biol. Chem. 263: 2238-2244
Liu, Q. Y.,Karpinski, E., Pang, P. K. T. (1991) Tetrandrine inhibits both T and L calcium channel currents in ventricular cells. Abstract Book of 5th International Symposium on Calcium Antagonists: Pharmacology and Clinical Research, p. 94 Savineau,J. P., Mironneau, J. (1990) Caffeine acting on pregnant rat myometrium: analysis of its relaxant action and its failure to release Ca from intracellular stores. Br. J. Pharmacol. 99: 261-267 Villar, A., DOcon, P., Anselmi, E. (1986) Role of intracellula calcium stores in the contractile response of uterus to several agonists. J. Pharmacol. 17: 541-546
